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Hi All,
Is there anybody had the experience that the singal of the analyte of interest are keeping decreased, while the internal standard kept stable. We use SHIMADZU LC20AD/API 3200, ESI+, mobile phase: ACN with formic acid (A)and water (B).
The compound has bad water solubility, if we ues UV dector, the signal was stable, however in LC/MS/MS, after repeated of some injections even neat solution. We thought there might be some compound itself or side-reaction during sythesis were dipot in the column, since after we wash the column, the signals increased. Many mobile phase/columns(C8,C18) /source(APCI, ESI-)were tried, considering the mass, we did not have too much choice for the acid or base to add in the MB. What's the next step we can try, UV? I'm afradi the sensitivity is not good enough. Change the MB? modify the gradent?
Please give me some suggestion? Thanks
Ning
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Hi, Ning:
Did you monitor the signal with MS and UV simultaneously? What kind of magnitude of the decrease within the day, 10-20%, 50%? Since UV is giving you stable signal, you can test using UV to see whether the compound is stable (from day to day) in the solution that you have been using. Different solvent solution might give different result. There are limited choices for mobile phase and reconstitution solutions for MS/MS, but you can still try a few. Especially with reconstitution solution, I would suggest you try buffer (e.g. ammonium acetate with desired PH value). I have dealt with some compound that decrease in certain solution, but more stable in buffered solution.
Another thought i have is that can you try isocratic run? Would it be possible that the compound has such poor solubility and changing the gradient causes change in the response? Does the decreasing you see happening the same to the high and low concentration samples?
If it is as you suggested that there is reactions going on between the column and the compound, then you would see UV signal decreasing as well.
Good luck,
Xiaohong
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Hi
We had a similar problem last year with the same instrument as yours (Shimadzu HPLC and AB Sciex 3200). Interestingly, the signal was only decreasing for the compound of interest (tacrolimus in our case) than any other compounds tested. Then it transpired that our instrument was "charging" so the mass spect engineer had to open the machine and clean first and third quads to resolve the problem.
Regards
Fatemeh Akhlaghi
Uni. of Rhode Island
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The following message was posted to: PharmPK
Hi all,
I agree with Dr. Fatemeh's comments.It can be by charging or contamination in Quad 1 & 3. Check by changing the ion source, whether you have similar observation ( ESI to APCI-if sensitivity permits. My guess, as you compound is less soluble in water, APCI may help relatively non-polar compound !)
Did you ramp mass parameters again, please check for any mass shift in the analyte's mass range. Alternatively the same compound can be tested on other LC-MS/MS, to confirm whether the problem is compound specific or instrument related.
Hope it helps.
Regards, Ravi Kumar Trivedi, Ph.D.
Biocon-Bristol Myers Squibb Research Centre.
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The following message was posted to: PharmPK
If you are working in positive mode just switch over the instrument to
negative and then to positive mode. Initial injection shows very high
response and the response starts decreasing from consecutive
injections. This is one of the indication for charging in Quads. more
likely this will have impact on both analyte/s and internal standard
response. Alternatively to confirm this as per Dr. Trivedi the same
can be tested in other MS/MS.
Regards,
Chandramohan
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Hi
This is out of curiosity did you check the stock and spiking solution stability.
Is the molecule completely soluble in the reconstitution solution since you are expressing that the compound has poor solubility in water.
What is the nature of the compound weakly acidic or basic, based on this we can select the reconstitution solution which can keep the molecule stable and more soluble? Keep the organic content of reconstitution solution at higher concentration (10:90) water to organic content. Adjust the ph of the reconstitution solution based on the pka of the molecule. Finally keep the auto-sampler temperature between 2-8*C.
Hope this will help you out in solving your problem.
Please keep everybody updated if this works.
If you still face problems you can reach me at Harsha.Narasimhaiah.aaa.syngeneintl.com
Thanks and regards
Harsha.K.N
DMPK-BIOLOGY
SYNGENE-BIOCON
INDIA.
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